Archives Volume 20 No. 2, 2020 pp. 9693-9700 e-ISSN:2581-6063 (online), ISSN:0972-5210

GENETIC DIVERSITY AMONG AND DULCIS GENOTYPES USING RAPD AND ISSR MOLECULAR MARKERS

Mohamed H. El-Sheikh1, Rehab Y. Ghareeb2 and Amira A. Ibrahim2* and Elsayed E. Hafez2 1Plant production Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications (SRTA- City), Universities and Research Center District, New Borg El-Arab, Alexandria, Egypt. 2*Plant Protection and Biomolecular Diagnosis Department, Arid Lands Cultivation Research Institute, City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab, Alexandria, Egypt.

Abstract Genetic variability among one genotype of (Juglans regia L.) and three genotypes of (Prunus dulcis L.) were studied by using molecular characterization. Two markers generated variability among studied cultivars RAPD & ISSR. RAPD marker produce a total number of bands 63 consists of 27 monomorphic and 36 polymorphic bands, generated polymorphism as 57.14%. Among the resulted polymorphic bands, a13 unique bands were observed. Whenever, ISSR marker generates 68 total bands with 17 monomorphic bands and 51 polymorphic bands, from these polymorphic there were 17 unique bands. Obviously, ISSR gave polymorphism among the examined with percentage of 75%. Data of molecular attributes computed and illustrated highest similarity coefficient among hard and scrub almond genotypes (sweet almond) and also was represented in one cluster separated the studied 4 genotypes into two groups; one group includes walnut genotype and other group includes; sweet almond (scrub hard) and bitter almond. ISSR marker produces similarity of 75% higher than the RAPD marker 57.14%, so, ISSR molecular markers could be the suitable method to differentiate among walnut and almond genotypes rather than RAPD markers. Key words: Genetic polymorphism, Juglans regia, Prunus dulcis, RAPD-PCR and ISSR– PCR

Introduction family , it is cultivated native to the Middle Cultivated Juglans regia L. is a monoecious and East and South Asia (Colic et al., 2012). It well known dichogamous species, considered as the most edible nuts that, Almond distributed in subtropical Mediterranean and economic species in family Juglandaceae and called climate therefore, the almond production is concentrated as Persian walnut (Tabasi et al., 2020). Persian walnut in some regions; Mediterranean and Asian countries is wildly distributed all over the tropical and temperate (Simsek and Demirkiran, 2010). Moreover, Almond regions of the world from Americans and Europe to Asia characterized by its resistance to salinity and drought, so (APG IV, 2016). Its fame due to the highest nutritive farmers prefer to cultivate it in the most cultivated areas value that contain and rich in , minerals and , in the world (Sorkheh et al., 2011). in addition to it considered as one of the most natural Economic importance to almond due to its highest source of energy (Pereira et al. 2008). It has an effective content from and fatty acid. Total dry weight of almond effect on human health derived from its high antioxidant contains a percentage of oil varied from 48% to 67%. capacity, high concentration of fatty acids, as well as Their composition varied significantly from genotype to various of group B (Anderson et al., 2001). Oil another intra the same species. These includes both prepared from the walnut is immensely beneficial for of fatty acids and linoleic acids that considered the major women suffering from menstrual dysfunction (Ros and components in the oil, vartion from 12% to 27% was Mataix, 2006). recorded with fatty acids but it was ranged from 63% to The almond; Prunus dulcis L. (Batsch) belong to 78% oleic acids (Kodad and Socias, 2008). *Author for correspondence : E-mail : [email protected] Genetic variation for certain species is the key for 9694 Mohamed H. El-Sheikh et al. knowledge of management and future using of this species Table 2: Number of total bands, polymorphic bands and and/or its germplasm in plant breeding resulting in newly percentage of polymorphism of each primer cultivars (Bernard et al., 2018). Walnut and almond are generated. highly different in morphological and taxonomical features. Parameter RAPD ISSR So, studying the evaluation of genetic diversity among Studied taxa 4 4 different species based on; phenotypic and genotypic, No. of primers 6 6 so, molecular markers could be used to give definite Marker range (bp) 130-1300 100-1300 information about their genetic routes of each individual Total bands 63 68 in the same species (Ebrahimi et al., 2011). Actually, Monomorphic bands 27 17 morphological traits only not sufficient for accurate Polymorphic bands 36 51 identification of specific plant species (Kumar, 1999; No. of unique bands 13 17 Mahmoodi et al., 2013), morphological traits should be % Polymorphism 57.14% 75% confirmed by molecular characterization which resulted management and breeding for these important crops. in well classification and identification. The DNA markers are the efficient way has been suggested for the Materials and methods illustration of genetic variability and similarity within and Extraction of DNA among the genotypes (Kumar et al., 2019). There are Young of one genotype of walnut (Juglans many molecular markers were used to differentiate among the walnut and almond genotypes such as regia L.) and three genotypes of almond Prunus dulcis isozymes (Viruel et al., 1995), RAPD (Gouta et al., L. (Batsch); one genotype of bitter almond and two types of sweet ; scrub Almond and hard almond were 2008), ISSR (Martins et al., 2003), RFLP (Tabasi et al., 2020; Wang et al., 2017), AFLP (Martins et al., 2001) extracted using CTAB buffer (Kriz¡man et al., 2006) and SNP (Wu et al., 2008). The common molecular and the resulted DNA was purified and both of purity and concentration of the extracted DNA were determined marker was used in genetic variation among was ISSR (Mahmoud and Abd El-Fatah, 2020). using Nano drop. Zhaobin et al., (2016) studied the biodiversity among PCR Reaction four Chinese wild almonds and two cultivars of Six primers of RAPD and six primers of ISSR were Amygdalus communis L. using SRAP marker. Studies used to study the genetic diversity; their name, bands of the genetic genetic polymorphism among different size and sequences were shown in table 1. In the PCR, cultivars of walnut and almond were little. Due to the reaction mixture was 20µl containing 10µl of 2x TOP low number of the articles studied the genetic simpleTM DyeMIX-HOT, 5µl of each Primer, 1µl of polymorphism among the walnut and almond; we aimed genomic DNA and volume was completed into 20 µl with to study the genetic diversity among different genotypes sterile . The PCR schedule as; one cycle at 95ºC of almond and of walnut using the molecular maekers for 10min followed by 40 cycles of; 94ºC for 30 sec, RAPD-PCR and ISSR-PCR. This type of study will be 30ºC for 1min for each primer, 72ºC for 2min and a final important clues for plant species conservation and incubation at 72ºC for 5min. The PCR products were Table 1: List of RAPD and ISSR primers name, size ranges and their sequences. separated on a 1.5% agarose gel and the gel Marker Primer Sequence Size was visualized using DNA gel documentation name Range(bp) according to (Mandal et al., 2014). OP-A2 5´ GTG ATC GCA G 3‘ 200-500 Data analysis OP-A07 5´ GAA AGG GGT G 3‘ 130-500 All gels were photographed and analyzed RAPD OP- B7 5´ GGT GAC GCA G 3‘ 270-1300 using Bio-Rad video documentation system, OP-B11 5‘ GTA GAC CCG T 3‘ 250-1300 Model Gel Doc 2000. Only distinct, - - OP-C04 5 -CCG CAT CTA C- 3 280-900 reproducible, well-resolved fragments were OP-C9 5´ CTC ACC GTC C 3‘ 250-1300 scored as present (1) or absent (0) for each 14A 5 CTC TCT CTC TCT CTC TG 3‘ 100-800 of the RAPD and ISSR markers. 49A 5‘CAC ACA CAC ACA CAC AG 3‘ 400-1100 Dendrogram of cluster analysis and genetic ISSR HB-9 5‘ CAC ACA CAC ACA CAC A (AG) T 3‘ 150-1300 similarity were used to illustrate and estimate HB-10 5‘ (GAG) (AGA) TGC CC3‘ 150-1700 2 2 the genetic distances and relationships among HB-12 5‘ TCT CTC TCT CTC TCT CA 3‘ 100-1000 studied genotypes using the SYSTAT version HB-15 5´ GTG GTG GTG GC 3‘ 270-900 Genetic diversity among Juglans regia and Prunus dulcis genotypes using RAPD and ISSR molecular markers 9695

7.0 software (Wilkinso, 1997). lowest number of total bands as six bands with three monomorphic bands and three polymorphic bands as Results illustrated in table 2. Primers OPB7 and OPC4 gave For RAPD analysis highest number of unique bands as four bands. OPB7 Six primers were used with molecular size ranged produced three unique bands for bitter almond at size from 130-1300bp, about 63 total bands were generated 600, 1000 & 1300 bp. and one band unique characteristic with 27 monomorphic bands and 36 polymorphic bands for sweet almond (hard almond) at size 350 bp. Primer as shown in table 3. Primer OPC9 produced highest OPC4 generated three unique bands characteristic for number of total bands as 18 with 11 monomorphic bands walnut at sizes 280, 300 & 400 bp and one unique band and seven polymorphic bands. Primer OPA2 showed for sweet almond genotype (scrub almond). Regarding,

Plate 1: RAPD-PCR product profiles of walnut and almond genotypes. M: DNA marker. 1: walnut. 2: sweet almond (scrub). 3: sweet almond (hard). 4: bitter almond. 9696 Mohamed H. El-Sheikh et al.

Table 3: Bands characteristics produced by molecular markers (RAPD& ISSR) in walnut and almond genotypes. Marker Primer Total Monomor- Polymor- %Polym- name bands phic bands phic Bands orphism RAPD OP-A2 6 3 3 50% OP-A07 8 4 4 50% OP- B7 11 1 10 90.91% OP-B11 9 4 5 55.56% OP-C04 11 4 7 63.64% OP-C9 18 11 7 38.89% ISSR 14A 10 2 8 80% 49A 6 2 4 66.67% HB-9 11 4 7 63.64% HB-10 16 2 14 87.5% HB-12 13 3 10 76.92% Fig. 1: Cluster analysis showing the relationships between HB-15 12 4 8 66.67% walnut and almond genotypes by Distance metric is for OPC9 primer produced two unique bands. One band Euclidean distance Average linkage method using RAPD marker. A: walnut. B: sweet almond (scrub). C: characteristic for almond genotype at 850bp, while other sweet almond (hard). D: bitter almond. unique band specific for bitter almond genotype at 400 bp. Primer OPA2 gave one unique band for bitter almond at 500bp, while OPA7 produce one band characteristic for walnut genotype as unique band at 500 bp and primer OPB11 generated one unique band for walnut genotype at 400bp as illustrated in Plate 1.Higest percentage of polymorphism produced by RAPD primers was generated by primer OPC9 as 38.89%, while the lowest percentage of polymorphism produced by OPB7 as Table 4: Similarity coefficient between walnut and almond genotypes using RAPD marker. Genotypes 1 2 3 4 1 1 2 0.083 1 3 0.071 0.832 1 4 0.351 0.066 0.12 1 Fig. 3: Cluster analysis showing the relationships between walnut and almond genotypes by Distance metric is Table 5: Similarity coefficient between walnut and almond Euclidean distance Average linkage method using genotypes using ISSR marker. RAPD & ISSR markers. A: walnut. B: sweet almond Genotypes 1 2 3 4 (scrub). C: sweet almond (hard). D: bitter almond. 1 1 90.91% as shown in table 2. 2 -0.077 1 For ISSR analysis 3 -0.156 0.864 1 Six ISSR primers were used to study genetic diversity 4 -0.317 0.339 0.329 1 among almond and walnut genotypes produced 68 total Table 6: Similarity coefficient between walnut and almond bands with 17 total bands and 51 polymorphic bands with genotypes using RAPD and ISSR markers. molecular size varied from 100 to 1300 bp as appeared in Genotypes 1 2 3 4 table 3. ISSR-PCR product profiles gel showed in Plate 1 1 2. HB10 primer showed highest number of total bands 2 -0.003 1 as 16 bands with two monomorphic bands and 14 3 -0.052 0.85 1 polymorphic bands. Primer 49A generated lowest number 4 -0.011 0.219 0.238 1 of total bands as six bands with two monomorphic bands 1: walnut. 2: sweet almond (scrub). 3: sweet almond (hard). 4: and four polymorphic bands. Primer HB10 produced bitter almond. highest percentage of polymorphism among ISSR primers Genetic diversity among Juglans regia and Prunus dulcis genotypes using RAPD and ISSR molecular markers 9697 as 87.5%, where HB9 primer generated lowest for each primer characteristic for walnut genotype at percentage of polymorphism as 63.64% as recorded in molecular size of 800 & 1000 bp for HB12 primer and table 2. Primer HB10 generated highest number of unique molecular size of 800 & 900 bp for Hb15 primer. Primer bands as six bands, four unique bands for bitter almond 14 A produced one unique band for walnut genotype at at size bands 150, 190, 250 and 350 bp and two unique 800 bp and one unique band for sweet almond (hard bands characteristic for walnut genotype at bands with almond) at 100 bp. Primer 49 A produced one unique molecular size of 1600 and 1700 bp. band characteristic for bitter almond at 1100 bp and one Primer HB9 generated three unique bands specific unique band for walnut genotype at 700 bp as illustrated for walnut genotypes at molecular size of 400, 1200 and in gel profile in Plate 2. Comparative percentage of 1300 bp. Primers HB12 and HB15 gave two unique bands polymorphism among RAPD and ISSR molecular marker

Plate 2: ISSR-PCR product profiles of walnut and almond genotypes. M: DNA marker. 1: walnut. 2: sweet almond (scrub). 3: sweet almond (hard). 4: bitter almond. 9698 Mohamed H. El-Sheikh et al.

subgroup includes only bitter almond. Genetic distance similarity based on RAPD and ISSR primers was presented in table 6. Highest similarity was 0.85 between sweet almond (hard) genotype and sweet almond (scrub) genotype. Lowest similarity was -0.003 between walnut genotype and sweet almond genotype (scrub). Discussion Improving the species and production of new lines and cultivars are in relation with studying genetic diversity and species distribution that produce valuable information to breeding programs and conservation of genetic resources which associated with food production in agriculture (Tabasi et al., 2020). Many farmers cultivated Fig. 2: Cluster analysis showing the relationships between different cultivars and lines from walnut and almond in walnut and almond genotypes by Distance metric is temperate regions due to their nutritive importance and Euclidean distance Average linkage method using economical value, so these types of fruits considered as ISSR marker. A: walnut. B: sweet almond (scrub). C: important fruit . Estimation of genetic diversity for sweet almond (hard). D: bitter almond. both wild and cultivated plants depends on molecular was recorded in table 3, showed highest polymorphism markers with different ploidy levels (Aliyev et al., 2007). for ISSR primers was 75%. Evaluating phylogenetic relationships among studied Data analysis species and genetic fingerprinting by using molecular markers as RAPD and ISSR are efficient tools in Cluster analysis was conducted based on RAPD conservation and breeding programs (Mahmoodi et al., analysis, generated dendrogram divided into two groups 2012). Six RAPD primers analysis produced 63 total first group includes walnut genotype and bitter almond amplified bands and percentage of polymorphism was genotype, where the second group includes sweet almond 57.14% was higher than percentage of polymorphism (hard and scrub almond) as presented in Fig. 1. produced by RAPD marker studied by Fakhraddin et On the other hand similarity distance showed highest al., (2013) was observed in walnut using RAPD marker. similarity was 0.832 between sweet almond (hard) and The 63 total bands with 27 common bands and 36 sweet almond (scrub), while the lowest similarity was polymorphic bands were higher than the result of Nicese 0.066 among sweet almond (scrub) and bitter almond as et al., (1998) and Potter et al., (2002). represented in table 4. ISSR molecular marker are helpful in fields of genetic Cluster analysis was conducted to show genetic diversity, phylogenetic studies, gene tagging, genome diversity among studied genotypes based on ISSR primers mapping and evolutionary biology in a wide range of plant produced dendrogram divided into two groups, first group species (Jabbarzadeh et al., 2010). They have been include walnut genotype only, second group includes effectively used in many tree species, including walnut almond genotypes (sweet and bitter) in Fig. 2. (J. regia L.) (Pollegioni et al., 2003), olive (Olea Similarity genetic distance based on ISSR primers europaea L.) (Terzopoulos et al., 2005), mulberry was highest 0.864 between sweet almond (hard) and (Morus L.) (Vijayan et al., 2006) and (Prunus L. sweet almond (scrub), while the lowest was -0.077 spp.) (Liu et al., 2007). For ISSR marker, six primers between walnut genotype and sweet almond (scrub) as produced 68 total bands with 17 monomorphic bands and illustrated in table 5. 51 polymorphic bands illustrated genetic diversity among Cluster analysis generated dendrogram based on walnut and almond genotypes. This result in agreement RAPD and ISSR molecular markers was illustrated in with Chatti et al., (2010) who studied genetic variability Fig. 3 separated unto two groups, one group contains among Tunisian Ficus tree cultivars using 48 ISSR walnut genotype only, second group contains sweet markers and detect polymorphism among them. ISSR almond and bitter almond, where the second group also marker generated 17 unique bands among almond and divided into two subgroups, one subgroup includes two walnut genotypes was lower than unique bands (19) genotypes of sweet almond (scrub & hard) and other generated from nine ISSR were used to study genetic Genetic diversity among Juglans regia and Prunus dulcis genotypes using RAPD and ISSR molecular markers 9699 diversity among 18 almond genotypes (Abodoma et al., Walnut Genotypes (Juglans regia L.) in Sulaimani Region 2017). Cluster dendrogram in addition to similarity Using RAPD and SSR Molecular Markers. Jordan Journal correlation among walnut and almond genotypes of Agricultural Sciences, 9(3):. separated studied genotypes into two groups one group Fakhraddin, M.H.S., A.R.T. Nawroz and M.F. Jamal. Assessment and has highest similarity among two sweet almond of genetic relationship among some iraqi walnut genotypes genotypes (hard & scrub) and other group with lowest (Juglans regia L.) in Sulaimani Region Using RAPD and similarity between walnut and sweet almond genotypes SSR Molecular Markers. J.J.A.S., 9: 351-362. in agreement with the result of Shah et al., (2019). ISSR Gouta, H., E. Ksia, N. Zoghlami, M. Zarrouk and A. Mliki (2008). marker produces higher similarity 75% than RAPD Genetic diversity and phylogenetic relationships among marker 57.14%, so ISSR molecular markers gave Tunisian almond cultivars revealed by RAPD markers. Journal of Horticultural Science and Biotechnology, 83: efficient result in diversity among species than RAPD 707-712. markers with regards to polymorphism detection using in genetic diversity between walnut and different almond APG VI (2016). An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering genotypes. This validates that ISSR markers are useful plants. Bot. J. Linn. Soc., 181: 1–20. markers in genetic divergence studies allowing an Jabbarzadeh, Z., M. Khosh-khui, H. Salehi and A. Saberivand unequivocal identification for genotypes. The high level (2010). Inter simple sequence repeat (ISSR) markers as of polymorphism possibly reflects the outcrossing reproducible and specific tools for genetic diversity character of walnut because almost similar results have analysis of rose species. African Journal of been obtained in fruit and nut tree species like Biotechnology, 9(37): 6091-6095. (Ji et al., 2014). Ji, A., Y. Wang, G. Wu, W. Wu and H. Yang (2014). Genetic References diversity and population structure of North China mountain walnut revealed by ISSR. A.J.P.S., 5: 3194-3202. Abodoma, A.F., M.M. Shehata, A.A. EL-Sherif, M.H. Ammar Kodad, O., G. Estopañán, T. Juan, I. Socias and R. Company and K.H. Khar (2017). Biodiversity assessment for some (2009). Xenia effects on oil content and fatty acid and Almond genotypes cultivated in using SRAP and tocopherol concentrations in autogamous almond ISSR Egypt. J. Genet. Cytol., 46: 409-431. cultivars. J. Agric. Food Chem., 57(22): 10809-10813. Aliyev, R.T., M.A. Abbasov and A.C. Mammadov (2007). Krizman, M., D. Baricevic and M. Prosek (2006). Fast Genetic identification of diploid and tetraploid wheat quantitative determination of volatile constituents in species with RAPD markers. Turk J. Biol., 31(3): 173–180. fennel by headspace-gas chromatography. Analytica Anderson, K.J., S.S. Teuber, A. Gobeille, P. Cremin, A.L. Chimica Acta, 557: 267-271. Waterhouse and F.M. Steinberg (2001). Walnut Kumar, L.S. (1999). DNA markers in plant improvement. polyphenolics inhibit in vitro human plasma and LDL Biotechnol. Adv., 17: 143–183. oxidation. J. Nutr., 131: 2837–2842. Kumar, M., V. Rakesh Sharma, V. Kumar, U. Sirohi, V. Chaudhary, Bernard, A., T. Barreneche, F. Lheureux and E. Dirlewanger S. Sharma, G. Saripalli, R.K. Naresh, H.K. Yadav and S. (2018). Analysis of genetic diversity and structure in a Sharma (2019). Genetic diversity and population structure worldwide walnut (Juglans regia L.) germplasm using SSR analysis of Indian (Allium sativum L.) markers. PLoS One, 13(11): e0208021. collection using SSR markers. Physiology and molecular Chatti, K., G. Baraket, A. Ben Abdelkrim, O. Saddoud, M. , biology of plants: an international journal of functional M. Trifi and A. Salhi Hannachi (2010). Development of plant biology, 25(2): 377–386. molecular tools for characterization and genetic diversity Liu, W., S. Li, A. Zhang and D. Liu (2007). Genetic diversity analysis in Tunisian fig (Ficus carica) cultivars. Biochem. revealed by RAPD markers in plum collection of China. Genet., 48: 789-806. Acta Hortic., 734: 287-294. Colic, S., V. Rakonjac, G. Zec, D. Nikolic and M.F. Aksic (2012). Mahmoodi, R., F. Rahman and R. Paktarmani (2012). Genetic Morphological and biochemical evaluation of selected Diversity of Persian Walnut from as Revealed by Inter- almond [Prunus dulcis (Mill.) D.A.Webb] genotypes in Simple Sequence Repeat (ISSR) Markers. Journal- northern Serbia. Turkish Journal Agriculture and American Pomological Society, 66(2): 101-106. Forestry, 36: 429-438. Mahmoodi, R., F. Rahmani and R. Rezaee (2013). Genetic Ebrahimi, A., R. Fatahi and Z. Zamani (2011). Analysis of genetic diversity among Juglans regia L. genotypes assessed by diversity among some Persian walnut genotypes (Juglans morphological traits and microsatellite markers. Span J. regia L.) using morphological traits and SSRs markers. Agric Res., 11(2): 431–437. Sci Hortic., 130: 146–51. Mahmoud, A.F. and B.E.S. Abd El-Fatah (2020). Genetic Fakhraddin, M.H.S., A.T. Nawroz and M.F. Jamal (2013). Diversity Studies and Identification of Molecular and Assessment of Genetic Relationship among Some Iraqi 9700 Mohamed H. El-Sheikh et al.

Biochemical Markers Associated with Fusarium Wilt almond genotypesin Diyarbakir central district. Resistance in Cultivated Faba Bean (Vicia faba). Plant Agricultural Journal, 5(3): 173-180. Pathol J., 36(1): 11-28. Sorkheh, K., B. Shiran, M. Khodambshi, V. Rouhi and S. Ercisli Mandal, P.K., M. Kochu Babu, M. Jayanthi and V. Satyavani (2011). In vitro assay of native almond species (Prunus L. (2014). PCR based early detection of Ganoderma sp. spp.) for drought tolerance. Plant Cell Tissue and Organ causing basal stem rot of oil palm in India. Journal of Culture, 105(3): 395-404. Plantation Crops, 42(3): 392-394. Tabasi, M., M. Sheidai and D. Hassani (2020). DNA Martins, M., A. Farinha, E. Ferreira, V. Cordeiro, A. Monteiro, fingerprinting and genetic diversity analysis with SCoT R. Tenreiro and M. Oliveira (2001). Molecular analysis of markers of Persian walnut populations (Juglans regia L.) the genetic variability of Portuguese almond collections. in Iran. Genet Resour Crop Evol., 67: 1437–1447. Acta Horticulturae, 546: 449-452. Terzopoulos, P.J., B. Kolano, P.J. Bebeli, P.J. Kaltsikes and I. Martins, M., R. Tenreiro and M. Oliveira (2003). Genetic Metzidakis (2005). Identification of Olea europaea L. relatedness of Portuguese almond collection assessed by cultivars using inter-simple sequence repeat markers. Sci. RAPD and ISSR markers. Plant Cell Report, 22: 71-78. Hort., 105: 45-51. Nicese, F.P., J.I. Hormaza and G.H.McGranahan (1998). Molecular Vijayan, K., P.P. Srivastava, C.V. Nair, A. Tikader and A.K. characterization genetic relatedness among walnut Awasthi (2006). Molecular characterization and (Juglans regia L.) genotypes based on RAPD markers. identification of markers associated with yield traits in Euphytica., 101: 199-206. mulberry using ISSR markers. Plant Breed., 125: 298-301. Pereira, J.A., I. Oliveira, A. Sousa, I.C. Ferreira, Bento and L. Viruel, M.A., R. Messeguer, M.C. De Vicente, J. Garcia-Mas, P. Estevinho (2008). Bioactive properties and chemical Puigdomenech, F.J. Vargas and P. Arús (1995). A linkage composition of six walnut (Juglans regia L.) cultivars. map with RFLP and isozyme markers for almond. Theor. Food Chem. Toxicol., 46: 2103–2111. Appl. Genet., 91: 964-971. Pollegioni, P., S. Bartoli, F. Cannata and M.E. Malvolti (2003). Wang, X., L. Li, J. Zhao, F. Li, W. Guo and X. Chen (2017). Genetic differentiation of four Italian walnut (Juglans regia Effects of different preservation methods on inter simple L.) varieties by inter simple sequence repeat (ISSR). J. sequence repeat (ISSR) and random amplified polymorphic Genet. Breed., 57: 231-240. DNA (RAPD) molecular markers in botanic samples. C. R. Potter, D., F.Y. Gao, G. Aiello, C. Lesle and G. Mcgranahan (2002). Biol., 340(4): 204-213. Inter-simple sequence repeat markers for fingerprinting Wilkinson, L. (1997). SYSTAT: The system analysis for static and determining genetic relationships of walnut (Juglans SYSTAT, Evaston III. regia) cultivars. J. Amer. Soc. Hort. Sci., 127: 75-81. Wu, S.B., M.G. Wirthensohn, P. Hunt, J.P. Gibson and M. Ros, E. and J. Mataix (2006). Fatty acid composition of nuts– Sedgley (2008). High resolution melting analysis of almond implications for cardiovascular health. Br. J. Nutr., 96: SNPs derived from ESTs. Theor. Appl. Genet., 118: 1-14. S29–S35. Zhaobin, J., C. Jimin, G. Chunhui and X. Wang (2016). Shah, U.N., J.I. Mir, N. Ahmed and K.M. Fazili (2019). Genetic traits, nutrient elements and assessment of genetic Diversity Analysis of Walnut (Juglans regia L.) from diversity for almond (Amygdalus spp.) endangered to Kashmir Valley Using RAPD and ISSR Markers. China as revealed using SRAP markers. African J. Biotec., Agrotechnology, 8: 185. 49: 51-57. Simsek, M. and A.R. Demirkiran (2010). Determination of superior